Plant Pathogens
Plants are exposed to numerous denizens of their environment, including bacteria, viruses, fungi and nematodes. Although many of the interactions between these organisms and plants, particularly via the roots of the plants, are beneficial, many of the interactions are harmful to the plants. The decimation of agricultural crops, ornamental plants and other plants by diseases caused by plant pathogens, particularly bacterial pathogens, is a worldwide problem that has enormous economic impact.
There are many pathogenic species of bacteria, fungi and nematodes. Fungal plant pathogens include species from the genera Fusarium, Pythium, Phytophthora, Verticillium, Rhizoctonia, Macrophomina, and others (see, e.g., U.S. Pat. No. 4,940,840 to Suslow et al.). Diseases caused by such fungal species include pre- and post-emergence seedling damping-off, hypocotyl rots, root rots, crown rots, and others. Nematode pathogens, which include species from the genera Meloidogyne heterodera, and others, cause diseases such as root galls, root rot, stunting and various other rots. In addition, nematodes serve as vectors for viral plant pathogens.
Bacterial pathogens have a significant impact on worldwide agriculture. Such pathogens include species of Pseudomonas, Erwinia, Agrobacteria, Xanthomonas, Clavibacter and others. Pseudomonas and Xanthomonas species affect a large number of different crops. For example, Pseudomonas tolaasii Paine, which causes brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach, produces an extracellular toxin, tolaasin, which disrupts membranes of fungal, bacterial, plant and animal cells; Pseudomonas syringae pv. tomato causes bacterial speck on tomato; Xanthomonas campestris pv. malvacearum causes angular leaf spot on cotton; and Pseudomonas solanacearum causes bacterial wilt on potatoes. Potatoes are also susceptible to post-harvest soft rot diseases caused by Erwinia carotovora. Such post-harvest soft rot diseases caused by Erwinia carotovora subsp. carotovora have a substantial impact on the potato industry.
Agricultural production of major crops has always been impeded by plant pathogens. Often diseases caused by plant pathogens limit the growth of certain crops in specific geographical locations and can destroy entire crops. Crop losses resulting from the deleterious effects of plant pathogens are, thus, a serious worldwide agricultural problem, particularly since there are no known treatments for many of the diseases caused by plant pathogens. Even in instances in which agrichemicals and pesticides are effective, their use is increasingly under attack because of the deleterious effects on the environment and on those who handle such products.
Because pesticides are often ineffective, unavailable and environmentally unacceptable, there is a need to develop alternative means for effectively eradicating or reducing the harmful effects of plant pathogens. In recent years, research has focused on the development of means for biocontrol of such pathogens and on the development of pathogen-resistant plants by breeding or by genetic engineering. There are, however, few reports of successful production of disease-resistant plants.
U.S. Pat. No. 4,940,840 to Suslow et al., however, describes the production of recombinant strains of Rhizobacterium that express heterologous DNA encoding the bacterial enzyme chitinase, which degrades chitin. Chitin is an unbranched polysaccharide polymer that contains N-acetyl-D-glucosamine units and occurs as an integral part of the cell walls of fungi and the outer covering of nematodes, nematode eggs and nematode cysts. The chitinase-expressing bacteria are applied to the plants or to soil as a means for inhibiting the growth of chitinase-sensitive fungi and nematodes. U.S. Pat. No. 4,940,840 also describes the production of transgenic tobacco plants that express chitinase and that appear to express sufficient levels of chitinase to inhibit the growth of fungal pathogens and are thereby less susceptible to diseases caused by such pathogens. Bacterial pathogens, however, do not have chitinous cell walls and are not inhibited by chitinase.
Bacterial Cell Walls
Bacterial cell walls are composed of peptidoglycan, which is a polysaccharide that contains amino sugars that are covalently crosslinked via small peptide bridges. The basic recurring unit in the peptidoglycan structure is the muropeptide, which is a disaccharide of N-acetyl-D-glycosamine and N-acetylmuramic acid in .beta.(1.fwdarw.4) linkage. Tetrapeptide side chains, containing L-alanine, D-glutamic acid or D-glutamine, and either meso-diaminopimelic acid, L-lysine, L-hydroxylysine or ornithine, are attached to the carboxyl group of the N-acetylmuramic acid residues. The parallel polysaccharide chains are cross-linked through their peptide side chains. The terminal D-alanine residue of the side chain of one polysaccharide chain is joined covalently with the peptide side chain of an adjacent polysaccharide chain, either directly as in Escherichia coli (E. coli), or through a short connecting peptide as in Staphylococcus aureus. The peptidoglycan forms a completely continuous covalent structure around the cell.
The cell walls of both gram-positive and gram-negative bacteria include a peptidoglycan backbone. Gram-positive bacteria possess cell walls that contain multilayered peptidoglycan and are encased in up to 20 layers of cross-linked peptidoglycan. Gram-negative bacteria have cell walls that contain mono or bilayered peptidoglycan and are more complex than the cell walls of gram-positive bacteria. Gram-negative and gram-positive bacteria also differ in accessory components, which are attached to the peptidoglycan backbone. The accessory components include polypeptides, lipoproteins and complex lipopolysaccharide (LPS), which form an outer lipid membrane surrounding the peptidoglycan skeleton. In particular, the cell walls of gram-negative bacteria contain a very complex lipopolysaccharide, which forms an outer membrane that provides gram-negative bacteria with a unique barrier that reportedly functions to selectively exclude environmental molecules, including lysozyme (Hancock, R. E. W. (1991) ASM News 57:175-182).
Lysozymes
The peptidoglycan backbone of the bacterial cell wall is resistant to peptide-hydrolyzing enzymes, which do not cleave peptides that contain D-amino acids, but can be cleaved by lysozymes. Lysozymes are a ubiquitous family of enzymes that occur in many tissues and secretions of humans, other vertebrates and invertebrates, as well as in plants, bacteria and phage. Lysozymes, which are 1,4-.beta.-N-acetylmuramidases, are basic enzymes that catalyze the hydrolysis of the .beta.-(1-4)glycosidic bond between the C-1 of N-acetylmuramic acid and the C-4 of N-acetylglucosamine, which occurs in the component of bacterial cell walls, bacterial peptidoglycan or murein. Some lysozymes also display a more or less pronounced chitinase activity, corresponding to a random hydrolysis of 1,4-.beta.-N-acetyl-glucosamine linkages in chitin. A slight esterase activity of lysozymes has also been reported.
Types of lysozymes
Several different types of lysozymes, based upon their amino acid sequence and structure, have been identified.
Lysozymes of the c, or "chicken" type contain 129-130 amino acids in their mature, secreted forms. About 40 of the 129-130 amino acids appear to be invariant among different species. Two of the several carboxyl groups of the type c lysozymes, which correspond to the Glu-35 and Asp-52 of the chicken egg white lysozyme, occur in similar positions in all c-type lysozymes. These groups are essential for lysozyme activity. A third carboxyl group, which corresponds to Asp-101 in chicken egg white lysozyme, is involved in a substrate binding interaction, and occurs in most c-type lysozymes. The eight half-cysteine residues of all of the c-type lysozymes are invariant. The disulfide bonds formed by the cysteines play an important role in the formation and maintenance of the secondary and tertiary structures of the lysozymes, which appear to be similar for all type c lysozymes.
The complete primary structures are known for the mature lysozyme c from numerous sources, including (1) hen egg white, quail, turkey, guinea fowl, duck and pheasant; (2) human milk and urine; (3) moth; (4) baboon; (5) rat; and (6) bovine stomach. The sequence of DNA that encodes mature human milk lysozyme c is also known. See European Patent Application Publication Nos. 0 181 634, 0 208 472, and 0 222 366.
The g-type lysozymes contain about 185 amino acids in their mature forms, exhibit low activity on N-acetylglucosamine polymers, and do not cross-react immunologically with lysozymes of the c-type. Lysozymes of the g-type have an unusually high occurrence of paired amino acids, in which the same amino acid occurs at neighboring positions in the molecules, and all of the four half-cysteine residues in the g-type molecules are situated in the N-terminal half of the chain. C-type lysozymes are equally active on peptide-substituted or unsubstituted peptidoglycan and are also active on chitin oligosaccharides. G-type lysozymes, which have activity against the linear peptidoglycan similar to that of the c-type enzymes, do not act on chitin oligosaccharides. Furthermore, g-type enzymes act only as hydrolases, whereas, c-type lysozymes are capable of both hydrolysis and transglycosylation.
The existence of other distinct types of lysozymes, which differ from the c and g types on the basis of structural, catalytic and immunological criteria, has also been reported. Bacteriophage lysozymes, such as T2 and T4 phage lysozymes, include 164 amino acids and have a molecular weight of 18,700. A lysozyme activity has been detected in several plant tissues, but the plant lysozyme appears to act as a chitinase rather than as a 1,4-.beta.-N-acetylmuramidase.
In addition, lysozymes may also be characterized on the basis of their in vivo biological activity. Because of their ability to cleave the peptidoglycan bacterial cell wall, some lysozymes are involved in mammalian defense systems. In addition, since lysozymes also possess the ability to indirectly stimulate the production of antibodies against a variety of antigens, such enzymes may also be employed to enhance resistance against infection. Lysozymes may also have anti-tumor activity.
A number of mammalian species, which have a foregut or rumen, have an unusually high level of lysozyme in the fundic region (anterior part) of the abomasum (stomach). This lysozyme, which has a unique activity profile, appears to have evolved to perform functions distinct from other lysozymes. Most lysozymes appear to function to protect against infection and in other defense systems. Ruminant stomach lysozymes, however, have evolved as digestive enzymes to digest the microbes that grow in the foregut and thereby scavenge the nutrients used by these microbes that digest cellulose.
Ruminant Gut Lysozymes
A ruminant is a cud-chewing mammal with two stomachs: a foregut in which anaerobic gram-positive bacteria digest cellulose, thereby permitting the ruminant to use cellulose as a source of energy and nutrients; and a true stomach. Ruminants, such as domestic cattle and other cud-chewing mammals in the order Artiodactyla have developed a symbiotic relationship with bacteria that live in the rumen thereby permitting ruminants to use cellulose as a major nutrient. The bacteria digest cellulose and other dietary components and rapidly grow and divide to large numbers. They convert a significant percentage of the nutrients that are ingested by the ruminant. The bacteria then enter the fundic region of the stomach.
Cell walls of the bacterial cells are digested only slowly by the normal repertoire of enzymes that are present in the mammalian gut, but, concomitant with the evolution of the symbiotic relationship with microbes, lysozymes that digest the cell walls of the microbes under the acidic conditions in the true stomach of ruminants and ruminant-like species, including cows, sheep and deer, have evolved (Dobson et al. (1984) J. Biol. Chem. 259:11607-11616). The lysozymes digest the bacteria, thereby enabling the ruminant to utilize the lysed bacteria as a source of carbon, nitrogen, and phosphorus for energy and growth.
Bovine stomach lysozyme was first purified from abomasum mucosa by Dobson et al.. ((1984) J. Biol. Chem. 259:11607-11616). Three distinct, related, non-allelic forms of lysozyme c were isolated. The three forms of lysozyme constitute approximately 10% of the total protein that can be extracted from the abomasum mucosa. These three nonallelic lysozymes, designated c1, c2 and c3, are closely related to one another antigenically and in amino acid composition. These type c lysozymes have functionally diverged from other mammalian lysozymes in that (1) the pH optimum for their enzymatic activity is approximately 5, rather than 7 as exhibited by other type c lysozymes; and (2) the type c lysozymes present in bovine abomasum are more stable in acidic environments, such as that of the abomasum, and are more resistant to proteolytic enzymes, such as pepsin, which occur in the abomasum, than other type c lysozymes (see, Jolles et al. (1984) J. Biol. Chem. 259:11617-11625). In addition, the complete 129 amino acid sequence of a mature bovine lysozyme c2 and the observation that antibodies prepared against the stomach lysozyme do not cross-react with non-digestive lysozymes from other tissues and secretions indicate that this enzyme appears to have diverged from other lysozymes (Jolles (1984) J. Biol. Chem., 259:11617-11625).
Ruminant lysozyme c, thus, is a digestive enzyme that lyses foregut gram-positive bacteria in the stomach and the proximal part of the small intestine, which permits ruminants to use the lysed bacteria as sources of carbon, nitrogen and phosphorous. This lysozyme is confined to the stomach; it has not been found in other tissues or secretions. In non-ruminant species, stomach lysozymes appear to be identical with the lysozymes in other tissues and secretions.
Lysozymes as Anti-Bacterial Agents
Lysozymes are known to exhibit anti-bacterial activity and activity against other pathogens, such as nematodes, that contain chitin. In addition, when used as antimicrobial agents, lysozymes are generally employed in combination with other agents, such as lacto-transferrin, complement, antibodies, vitamins, other enzymes and various antibiotics, including tetracycline and bacitracin. Such antimicrobial compositions are used as preservatives for foods, such as cheese, sausage and marine products, as ripening agents for cheese, and also in skin, hair care and other cosmetic compositions.
Antimicrobial compositions that contain ruminant lysozyme c and endo-.beta.-N-acetylglucosaminidase or endoglycopeptidase that are formulated as mouthwashes, soaps, contact lens cleaners and other similar products are described in European Patent Application 0 42 019 A1 (Feb. 5, 1991, THE PROCTOR & GAMBLE COMPANY). These compositions include the endo-.beta.-N-acetylglucosaminidase or endoglycopeptidase in addition to the lysozyme because the lysozyme c is not sufficiently effective against bacteria, such as Staphylococcus aureus, that occur on the skin, and in the mouth and eyes, to be used alone.
In addition, lysozymes are purportedly not effective against gram-negative bacteria (see, European Patent Application 0 42 019 A1, Feb. 5, 1991, THE PROCTOR & GAMBLE COMPANY). As discussed above, gram-negative bacteria are encased in a lipopolysaccharide (LPS) outer membrane, which reportedly functions to exclude environmental molecules, including lysozyme (Hancock, R. E. W. (1991) ASM News 57:175-182).
Thus, lysozyme appears to be most useful, when used as an antimicrobial agent, for lysing gram-positive bacteria. It would not appear that lysozyme is the agent of choice for organisms such as plants that are plagued by diseases caused by gram-negative organisms.
The majority of bacterial plant pathogens, about 95%, including Agrobacterium tumefaciens, Pseudomonas syringae, Xanthomonas campestris and Erwinia carotovora, are gram-negative. In addition, as described in the examples below, lysozymes, such as chicken lysozyme, are unstable, particularly under conditions in which plants are grown or under which seeds are stored. Therefore, it would appear that lysozymes are unsuitable for treating plants, since most bacterial pathogens are gram-negative and lysozymes are too unstable to protect against infection for sufficient time to be effective.
Control of Plant Pathogens
There are few effective treatments or means for preventing plant diseases of bacterial origin or for controlling plant bacterial pathogens. The treatments that are used are often environmentally unsound and generally do not have systemic or prophylactic activity. Pesticides, including heavy metal-containing sprays and antibiotics, such as streptomycin, are no longer considered environmentally acceptable and are often ineffective. For example, Pseudomonas syringae pv. tomato, which causes bacterial speck on tomato, is presently controlled by frequent application of copper-containing sprays, which, in addition to their unfavorable environmental impact, select for copper-resistant strains. Treatment of apple and pear orchards with streptomycin in order to control the blight pathogen, Erwinia amylovora, has resulted in the appearance of streptomycin-resistant strains. Xanthomonas campestris pv. malvacearum, which causes angular leaf spot on cotton, presently is only controlled by treating seeds with mercury-containing compounds and copper sprays. Other Xanthomonas campestris species, such as X. campestris pv. vesicatoria and X. campestris pv. campestris, can be seedborne, and there are no effective means for treating the seeds without injury.
Since there are few means for controlling plant bacterial pathogens, and those that are available, such as heavy metal-containing sprays and antibiotics, are not highly effective and are environmentally unacceptable, and since there are relatively few bacterial pathogen-resistant vegetable or fruit plants available, there is a need for the development of effective, non-toxic, biodegradable and environmentally acceptable means for the control of plant pathogens. There is also a need to develop disease-resistant plants and to develop means for treating plants to eradicate or control plant diseases of bacterial origin.
Therefore, it is an object of this invention to provide methods for treating plants, plant tissues, and seeds infected with plant pathogens and for treating non-infected plants to render them resistant to or less susceptible to infection by plant pathogens.
It is another object to provide methods for disinfecting seeds to render the seeds and resulting plants free from infection by common plant pathogens.
It is a further object to provide transgenic plants that are resistant to or less susceptible to infection by a variety of plant pathogens.
It is also an object to provide transgenic plants that express and properly process the product of a heterologous gene that encodes an agent that effectively inhibits the growth of or eradicates a variety of plant pathogens, and to provide a means for effecting the proper processing of the heterologous gene product.